Functionality and cell anchorage dependence of the African swine fever virus gene A179L, a viral bcl-2 homolog, in insect cells

Abstract

The African swine fever virus gene A179L has been shown to be a functional member of the ced9/bcl-2 family of apoptosis inhibitors in mammalian cell lines. In this work we have expressed the A179L gene product (p21) under the control of the baculovirus polyhedrin promoter using a baculovirus system. Expression of the A179L gene neither altered the baculovirus replication phenotype nor delayed the shutoff of cellular protein synthesis, but it extended the survival of the infected insect cells to very late times postinfection. The increase in cell survival rates correlated with a marked apoptosis reduction after baculovirus infection. Interestingly, prevention of apoptosis was observed when recombinant baculovirus infections were carried out in monolayer cell cultures but not when cells were infected in suspension, suggesting a cell anchorage dependence for p21 function in insect cells. Cell survival was enhanced under optimal conditions of cell attachment and cell-to-cell contact as provided by extracellular matrix components or poly-D-lysine. Since it was observed that cytoskeleton organization varied depending on culture conditions of insect cells (grown in monolayer versus grown in suspension), these results suggested that A179L might regulate apoptosis in insect cells only when the cytoskeletal support of intracellular signaling is maintained upon cell adhesion. Thus, cell shape and cytoskeleton status might allow variations in intracellular transduction of signals related to cell survival in virus-infected cells.

FIG. 1

Expression of the ASFV A179L gene by using a baculovirus system. (A) Western blot analysis of infected Sf9 cell extracts. Cell lysates from Sf9 cells synchronously infected either with the recombinant baculovirus BVA179L or with BVA179L-AS reacted at 72 h p.i. with a specific antiserum raised against the A179L gene product expressed in E. coli cells. (B) Immunofluorescence of BVA179L- or BVA179L-AS-infected insect cells in suspension, analyzed at 72 h p.i. by using the same serum against A179L stained with fluorescein isothiocyanate (FITC). Green cytoplasmic fluorescence was detected only in cells infected with BVA179L. The cell nucleus was contrasted with propidium iodide (PI) (right). (C) Growth curves of BVA179L and BVA179L-AS recombinant baculoviruses in Sf9 monolayer cell cultures. Extracellular virus obtained from culture supernatants at the indicated time points was titrated in a conventional plaque assay. Squares, BVA179L-AS; circles, BVA179L.

FIG. 2

Effect of A179L gene expression on the survival of baculovirus-infected Sf9 cells. Viability of insect cells grown in monolayers (A) or in suspension (B) and infected with BVA179L-AS (squares) or BVA179L (circles) at a MOI of 100 was determined by trypan blue exclusion at different times postinfection. Means and standard errors were calculated from three independent experiments. (C) A representative field of trypan blue-stained monolayer cultures of BVA179L- or BVA179L-AS-infected Sf9 cells at 144 h p.i. Original magnification, ×200.

FIG. 3

DNA fragmentation in infected Sf9 cells (MOI of 10). (A) Hoechst 33258 staining of BVA179L- or BVA179L-AS-infected Sf9 cells grown in plates at 144 h p.i. Original magnification, ×400. (B) ELISA quantitation of the DNA linked to histone proteins in the cytoplasmic fraction of apoptotic Sf9 cells infected in monolayer at different time points. Squares: BVA179L-AS-infected cells; circles, BVA179L-infected cells; triangles, mock-infected cells. Means and standard errors were calculated from three independent experiments. (C) Agarose gel electrophoresis of the internucleosomal DNA laddering detected in infected or mock-infected Sf9 cells in monolayer cultures at 96 and 144 h p.i. (left) or in suspension cultures at 96 h p.i. (right). M, molecular size markers.

FIG. 4

(A) Apoptosis induction of Sf9 cells (expressing p21) expressed as the ratio to that in BVA179L-AS-infected cells grown on different substrates to improve adhesiveness, measured by ELISA. The AI is given by the quotient (BVA179L-infected cells/BVA179L-AS-infected cells) of mean absorbance values at 405 nm from two replicate experiments at 96 h p.i. FN, human fibronectin; PDL, poly-d-lysine; COLI, rat tail collagen type I; UNTREATED, BVA179L-infected cells. (B) Actin cytoskeleton organization in adherent insect cells (left). Cells cultured in suspended growth conditions presented actin focalization (right). Cellular F-actin was stained with phalloidin-tetramethyl rhodamine isothiocyanate. (C) Characteristic individual infected Sf9 cells cultured in monolayer (upper) or in suspension (lower). Actin clumped in coarse fragments showed intense red staining. Original magnification, ×600 (panels B and C).